Effects of various diets on the nutritional value of Artemia sp. as food for the prawn Penaeus monodon

The nutritional value of Artemia sp. as food for marine fish and crustacean larvae has been linked to the level of its polyunsaturated fatty acid (PUFA) content. Experiments in August 1984 were conducted to determine the effects of various artificial diets and algae on fatty acid composition of PUFA-deficient Artemia sp. (Utah GSL strain) and their resulting value as food for postlarvae of the prawn Penaeus monodon (Fabricius). Nauplii of the brine shrimp were grown on extracts of corn, copra, soybean and rice bran containing precursors (C18) to long-chain PUFA and also on algal species containing different levels of long-chain PUFA (C20). The nauplii were then used as food for P. monodon postlarvae. The results revealed that absence of C20 polyunsaturates from the feeds and their presence in the algae were reflected in the polyunsaturated fatty acid content of the tissues of Artemia sp. When fed with brine shrimp fed on algae, P. monodon displayed better postlarval survival and significantly higher growth; related to the content of polyunsaturated fatty acids in Artemia sp. A practical feeding approach in prawn hatcheries would be to grow Artemia sp. on a cheap diet such as rice bran, and then to enhance its nutritional value with a diet high in PUFA prior to harvesting, in order to improve hatchery production.     

Relative importance of prey size and concentration in determining the feeding behaviour of the herringClupea harengus

The feeding behaviour ofClupea harengus L. in the light is dependent primarily on prey concentration. In the laboratory the fish feed by biting at low prey concentrations and by filtering at high concentrations. With the brine shrimpArtemia sp. as prey, the concentration required for the onset of filter-feeding was directly dependent on prey size, but the concentration at which 50% of feeding fish were filtering differed little between three sizes of brine shrimp (nauplii, and 2 and 4 mm larvae). When fed onCalanus finmarchicus, however, 50% of fish fed by filtering at concentrations at least six times lower than on any size of brine shrimp. Filter-feeding thresholds forC. finmarchicus were six to ten times lower than for any size ofArtemia sp. and, on the basis of biomass, approximately eight times lower than for equivalent sizedArtemia sp.     

Electrophoretic survey of genetic variation in Pecten maximus,Chlamys opercularis,C. varia and C. distorta from the Irish Sea

Food selection by young larvae of the gulf menhaden (Brevoortia patronus) was studied in the laboratory at Beaufort, North Carolina (USA) in 1982 and 1983; this species is especially interesting, since the larvae began feeding on phytoplankton as well as microzooplankton. When dinoflagellates (Prorocentrum micans), tintinnids (Favella sp.), and N1 nauplii of a copepod (Acartia tonsa) were presented to laboratory-reared, larval menhaden (3.9 to 4.2 mm notochord length), the fish larvae ate dinoflagellates and tintinnids, but not copepod nauplii. Larvae showed significant (P<0.001) selection for the tintinnids. Given the same mixture of food items, larger larvae (6.4 mm notochord length) ate copepod nauplii as well as the other food organisms. These feeding responses are consistent with larval feeding in the northern Gulf of Mexico, where gulf menhaden larvae between 3 and 5 mm in notochord length frequently ate large numbers of dinoflagellates (mostly P. micans and P. compressum) and tintinnids (mostly Favella sp.), but did not eat copepod nauplii. As larvae grew, copepod nauplii and other food organisms became important, while dinoflagellates and tintinnids became relatively less important in the diet. Since the tintinnids and nauplii used in the laboratory feeding experiments were similar in size as well as carbon and nitrogen contents, the feeding selectivity and dietary ontogeny that we observed were likely due to a combination of prey capturability and larval fish maturation and learning.Contribution No. 5575 of the Woods Hole Oceanographic Institution     

Characteristics of feeding on dinoflagellates by newly hatched larval crabs

The zoeal larvae of brachyuran crabs must feed soon after hatching on a diet that includes large micro- and mesozooplankton in order to satisfy nutritional requirements. However, newly hatched larvae have been shown to ingest a variety of dinoflagellates, perhaps using microbial carbon sources to sustain them until they encounter more favored prey. Ingestion of dinoflagellates by larval crabs has been documented previously under conditions in which the larvae were exposed to algae provided in monoculture or in defined mixtures of cells. We report here on experiments conducted on the hatching stage of five crab species to determine if ingestion of dinoflagellates occurred when they were provided in combination with Artemia sp. nauplii or after a period of feeding on mesozooplankton. Quantitative measurements of chl a in the larval guts provided evidence of ingestion of algal cells. Active ingestion of the dinoflagellate Prorocentrum micans at specified intervals during an extended feeding period was determined on larvae of two crab species using fluorescently labeled cells provided for brief periods at prescribed time intervals. Stage 1 larvae of four of the five crab species ingested dinoflagellates when they were provided in combination with nauplii and larvae of all five species ingested cells after feeding solely on nauplii for 24 h. Ingestion of algal cells was first evident in the larval guts after 6 h of feeding at both low (200 cell ml⁻¹) and high (1,000 cells ml⁻¹) prey densities. Higher prey densities resulted in higher gut chl a. Larvae continuously exposed to dinoflagellates actively ingested cells at every 3 h interval tested over a 36 h period. Results confirm previous studies that larvae will ingest dinoflagellates even when they are encountered in a mixed prey field or when having previously fed. Ingestion of cells may occur on a continual basis over time.     

Importance of the fatty acids 20:5ω 3 and 22:6ω 3 in the diet of plaice (Pleuronectes platessa) larvae

The requirement of plaice (Pleuronectes platessa L.) larvae for the fatty acids 20:5 3 and 22:6 3 was studied. Larvae were reared from first feeding to beyond metamorphosis on Artemia sp. nauplii (EG brand), whose nutritional content had been manipulated by enrichment. Some larvae were fed Artemia rich in 20:5 3 and 22:6 3, and others were fed nauplii with no 22:6 3 and low levels of 20:5 3 (1.9% of the total fatty acids). The differences in diet had no significant difference on growth or survival of the plaice larvae. The results indicate that the plaice larvae in this study did not require 22:6 3 in their diet, even when the levels of 20:5 3 were low.     

Effects of diet on fatty acid composition of body zones in larvae of the sea bass Dicentrarchus labrax: a chemometric study

Larvae of the sea bass Dicentrachus labrax were fed four Artemia sp. diets for 28 d. Three were nauplii enriched with emulsions of polyunsaturated fatty acids, and the fourth nauplii enriched with baker's yeast. At the end of the experimental period, the fatty acids of the bodies, heads and eyes of the larvae were analysed. A multivariate statistical method (discriminant analysis, DA) applied to the data revealed anatomical as well as dietary fatty acid pattern-discrimination. We propose here the use of discriminant analysis as a pattern-recognition method that will help to integrate the fatty acid information obtained in nutritional studies.     

Trypsin content in intestines of herring larvae,Clupea harengus,ingesting inert polystyrene spheres or live crustacea prey

In 1986, at the Danish Institute of Fisheries and Marine Research, Denmark, Clupea harengus L. larvae from three different herring stocks were offered either non-biodegradable polystyrene spheres, nauplii and copepodites of Acartia tonsa or Artemia ssp. nauplii. Ingestion of polystyrene spheres induced trypsin secretion to a higher level than in non-feeding fish. Larvae ingesting live food of the same width as the polystyrene spheres exhibited the highest trypsin content in the intestines. Mechanisms responsible for the regulation of pancreatic enzyme secretion are discussed.     

Laboratory rearing of the clupeid fish Harengula pensacolae from fertilized eggs

Pelagic eggs of the scaled sardine Harengula pensacolae (Goode and Bean), have been hatched and reared in the laboratory for the first time. Larvae were reared in two 75 l aquaria under constant illumination, at an average temperature of 26.2°C. Zooplankton collected in a 35 mesh net was fed to the newly hatched larvae, and the diet was supplemented later with Artemia salina nauplii and a pelleted food. Larvae hatched at 4 mm TL (total length), and metamorphosed about 25 days later at 25 to 30 mm TL. Survivors averaged 76 mm TL 100 days after hatching. Of the 500 incubated eggs, 2.8% survived until 20 days, after which no significant natural mortality occurred. Sources of natural mortality included starvation, a copepod parasite (Caligus sp.), and injuries from contact with the sides of the tank. Larvae began feeding at 4.5 mm TL on copepod nauplii averaging 62 in body width. Scaled sardines were photopositive throughout the larval stage.Contribution No. 149, Bureau of Commercial Fisheries Tropical Atlantic Biological Laboratory, Miami, Florida 33149, USA.     

Light intensity and the feeding behaviour of herring,Clupea harengus

An infra-red sensitive video-recording technique was used to study the effects of darkness and light intensities from 0.0001 to 270 photopic lx on the feeding behaviour of herring (Clupea harengus L.). When offered natural zooplankton, consisting of a mixture ofCalanus finmarchicus, Euchaeta norvegica, Oithona similis, Balanus sp. nauplii, and crustacean nauplii as prey, the fish fed by biting (snapping) at light intensities above a threshold of 0.001 lx and were size-selective, taking the larger organisms first. When fed on pure cultures of CaliforniaArtemia sp. nauplii (San Francisco Bay brand), the threshold light intensity was 0.01 lx. Swimming speed increased with increasing light intensity when the fish were actively feeding by biting. When the fish were filter-feeding on high densities ofArtemia sp. nauplii in the light, they continued to school and swimming speed was not related to light intensity.     

Prey selection by larval weakfish (Cynoscion regalis): the effects of prey size,speed, and abundance

We examined feeding by larval weakfish, Cynoscion regalis (Bloch and Schneider), in laboratory experiments conducted during the 1991 spawning season. under natural conditions weakfish larval development is ca. 3 wk, and we ran separate experiments with larvae of five different ages (5, 8, 11, 14, and 17 d post-hatching). We used two different size classes of rotifers (Brachionus plicatilis) and brine shrimp nauplii (Artemia sp.) as prey organisms. Contrary to results of previous research, weakfish larvae did not select prey based on size alone. When prey abundance was above 100 itemsl^-1 weakfish, larvae always chose large rotifers (length = 216 m) over small rotifers (length = 160 m). At 11 d post-hatching, larvae switched their diet from large rotifers to small brine shrimp nauplii (length = 449 m); however, when fed small rotifers and small brine shrimp nauplii the change in diet occurred at 14 d post-hatching. This pattern of selectivity was maintained in each larval age class. Early-stage larvae (5 and 8 d post-hatching) did not feed selectively when prey abundance was less than 100 itemsl^-1. Late-stage larvae (17 d post-hatching) fed selectively at abundances ranging from 10 to 10000 items^-1. Lwimming speeds of prey items, which ranged from 1 to 6 mms^-1, had no consistent effect on prey selection. These results suggest that weakfish larvae are able to feed selectively, that selectivity changes as larvae age, and that selectivity is also influenced by prey abundance.     

Feeding on intertidal microbial mats by postlarval tiger shrimp, <Emphasis Type="Italic">Penaeus semisulcatus</Emphasis> De Haan

A series of experiments investigated the potential role of microbial mats in nutrition of the early settlement stages of Penaeus semisulcatus. From 3 days post-metamorphosis, the microbial mat supported high growth and survival rates in postlarvae, equivalent to that supported by a control diet of Artemia nauplii and mussel. Examination of gut contents indicated that benthic postlarvae feed indiscriminately on the microbial mat. However, when postlarvae were fed separated size-fractions of the microbial mat, only the fraction containing a high concentration of infauna (mainly nematodes) was able to support the same growth as intact microbial mat. This appears to be due to the low nitrogen content (0.4–0.9 mmol g⁻¹) of the various size-fractions, compared to that of infauna (4.0 mmol g⁻¹). The stable isotope composition of the dietary size-fractions and postlarval shrimp tissue supports the hypothesis that the shrimp assimilated C and N primarily from the associated infauna. This may be due to selective feeding that is not apparent from stomach contents, due to rapid digestion of fauna soft tissues, or to differential assimilation of infaunal prey relative to other microbial mat components. The results demonstrate that microbial mats may support survival and growth in early-stage penaeid shrimp postlarvae on intertidal mud flats.     

Effects of exposure to toxic and non-toxic dinoflagellates on oxygen consumption and locomotion in stage 1 larvae of the crabs Cancer oregonensis and C. magister

Sublethal effects on larval crabs upon exposure to toxic dinoflagellates were examined in the laboratory in early 1999. Specifically, oxygen consumption rates and geotaxis responses were determined for stage 1 larvae of the crabs Cancer oregonensis (Dana) and C. magister Dana that were exposed to non-toxic (Alexandrium tamarense, strain 115) or toxic (A. fundyense, strain 1719) dinoflagellates or to freshly hatched nauplii of the brine shrimp Artemia sp. In C. oregonensis, larvae exposed to the toxic dinoflagellate showed reduced rates of oxygen consumption compared to those exposed to non-toxic dinoflagellates or brine shrimp nauplii. Larvae exposed to a filtrate of the non-toxic dinoflagellate showed no change in oxygen consumption, but a reduced rate when exposed to filtrate from the toxic alga at densities >5᎒² cells ml^-1. In C. magister, larvae exposed to the non-toxic A. tamarense or the toxic A. fundyense had reduced oxygen consumption rates. Larvae exposed to filtrates of non-toxic and toxic dinoflagellates had no change in oxygen consumption. In geotaxis tests, C. oregonensis larvae exposed for 1 day to the toxic A. fundyense reduced their level of locomotion compared to those exposed to non-toxic A. tamarense or to brine shrimp nauplii. C. magister larvae showed no change in activity after a 1-day exposure to the toxic A. fundyense. After a 4-day exposure to A. fundyense, C. magister larvae had much reduced locomotion. Reduced locomotory activity in larvae exposed to toxic algae is consistent with the changes in oxygen consumption rates. Responding to exposure to toxic algae by reducing locomotion may affect vertical migration in these negatively buoyant crab larvae, resulting in sinking below a toxic alga bloom, at least temporarily.     

Effects of water motion and prey behavior on zooplankton capture by two coral reef fishes

Water motion is an important factor affecting planktivory on coral reefs. The feeding behavior of two species of tube-dwelling coral reef fish (Chaenopsidae) was studied in still and turbulent water. One species of blenny, Acanthemblemaria spinosa , lives in holes higher above the reef surface and feeds mainly on calanoid copepods, while a second, A. aspera , lives closer to the reef surface, feeds mainly on harpacticoid copepods, and is exposed to less water motion than the first. In the laboratory, these two blenny species were video recorded attacking a calanoid copepod ( Acartia tonsa, evasive prey) and an anostracan branchiopod (nauplii of Artemia sp., passive prey). Whereas A. spinosa attacked with the same vigor in still and turbulent water, A. aspera modulated its attack with a more deliberate strike under still conditions than turbulent conditions. For both fish species combined, mean capture success when feeding on Artemia sp. was 100% in still water and dropped to 78% in turbulent water. In contrast, when feeding on Acartia tonsa, mean capture success was 21% in still water and rose to 56% in turbulent water. We hypothesize that, although turbulence reduces capture success by adding erratic movement to Artemia sp. (passive prey), it increases capture success of Acartia tonsa (evasive prey) by interfering with the hydrodynamic sensing of the approaching predator. These opposite effects of water motion increase the complexity of the predator-prey relationship as water motion varies spatially and temporally on structurally complex coral reefs. Some observations were consistent with A. aspera living in a lower energy benthic boundary layer as compared with A. spinosa: slower initial approach to prey, attack speeds modulated according to water velocity, and lower proportion of approaches that result in strikes in turbulent water.Communicated by P.W. Sammarco, Chauvin     

Feeding behaviour and prey size selection of gilthead seabream,Sparus aurata,larvae fed on inert and live food

Prey selection shortly after the onset of feeding by laboratory-reared gilthead seabream, Sparus aurata L., larvae was studied using larvae fed on two types of microcapsule (hard- and soft-walled) having diameters ranging from 25 to 300 m. Preferences between inert food and live prey (rotifers and Artemia sp. nauplii) were also studied. Seabream larvae were able to ingest inert food from first feeding. Larvae of all size classes ingested hard microcapsules with diameters in the range 25 to 250 m. However, larvae with a total length (TL) below 4 mm preferentially selected particles 25 to 50 m in diameter, larvae of TL 4 and 5 mm preferred particles 51 to 100 m in diameter, while larvae above 5 mm TL preferred particles 101 to 150 m in diameter. With soft microcapsules, larvae always preferred particles larger than in the previous case, and above 4.5 mm TL they preferentially selected particles 201 to 250 m in diameter. In addition, the gradual increase of preferred diameters with increasing TL was more pronounced when larvae were increasing TL was more pronounced when larvae were fed on soft particles. Mean values for prey width/mouth width ratios were approximately 0.24 and 0.30 when larvae were fed on hard-walled and soft-walled microcapsules, respectively, irrespective of the absolute value of larval length. When a mixed diet of live and inert food items was offered, live prey were always preferentially selected, even if the prey width/mouth width ratio was apparently not favourable. Only a physical constraint such as excessive prey width could counter this preference for living prey vs inert microcapsules. These results contribute to our knowledge in larval feeding behaviour, especially in the presence of inert food, and represent a fundamental step in developing prepared food for marine fish larvae.     

Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids

The lipid profiles of a few species of marine unicellular algae and yeast were studied with emphasis on fatty acids as part of a search for the nutritional value of plankton to the diet of marine fish larvae commonly used in marine hatcheries. The general proximate chemistry of rotifers was closely related to the proximate chemistry of the diet organism, exhibiting a higher content of protein and carbohydrate and a lower content of lipid. Major lipids in all algae, yeast and rotifers comprised mono-, di- and tri-glycerides and polar lipids. The algae Chaetoceros gracilis Schutt, Isochrysis galbana Parke and their respective algaefed rotifers exhibited higher amounts of neutral lipids, consisting mainly of cyclic and branched polyunsaturated components. Fatty acid composition of the algae was species-specific, with the highest ratio of polyethylenic to saturated and monoethylenic acid in I. galbana and Phaeodactylum tricornutum Bohlin, and the highest content (15%) of n-3 highly unsaturated fatty acids in Nannochloropsis salina and P. tricornutum. A closely mirrored distribution of the fatty acids, but with a lower amount of n-3 highly unsaturated fatty acids, was present in the respective algae-fed rotifers. Comparison of the fatty acid spectrum of Artemia sp. and Euterpina acutifrons grown in the laboratory on I. galbana with zooplankton samples of E. acutifrons and Oitona nana collected from the sea showed a higher concentration of docosahexaenoic acid (22:6 n-3) in the naturally collected sample. The results indicate that the efficacy of the food algae C. gracilis and I. galbana in increasing the survival of fish larvae in marine hatcheries is not obvious on the sole basis of fatty acid composition.     

Effects of salinity and dietary DHA (22:6n - 3) content on lipid composition and performance of Penaeus kerathurus postlarvae

A two-way ANOVA experiment was designed to study the effects of salinity and dietary docosahexaenoic acid (DHA; 22:6n-3) on lipid composition and performance of postlarvae from the marine shrimp Penaeus kerathurus (Forskål, 1775). Shrimp were reared from 1- to 8-d-old postlarvae at 35 and 25‰S with Kelko-enriched Artemia sp. [20.0 μg (n-3)?HUFA mg^?1 dry weight; 9.1 μg DHA mg^?1 dry weight] and nonenriched Artemia sp. [14.2 μg (n-3)?HUFA mg^?1 dry weight; 0.3 μg DHA mg^?1 dry weight]. Dietary DHA content did not affect either total length or survival but influenced the nutritional status represented by condition indices (triacylglycerol/total polar lipid and triacylglycerol/free cholesterol) of 8-d-old postlarvae at the end of the experiment. Culture salinity affected final total length and condition indices but did not show any effect on survival in the different experimental treatments. The interaction of dietary DHA and culture salinity was not significant for total length and survival but was significant for both condition indices used. P. kerathurus 8-d-old postlarvae showed better growth, survival and nutritional condition when reared at 35‰S and when fed on Kelko-enriched Artemia sp., but the differences with postlarvae from other treatments were very poorly marked. The results demonstrate that 8-d-old postlarvae may have sufficiently developed osmoregulatory capabilities to resist 25‰S under good conditions, although (n-3) HUFA-enriched diets may also enhance osmotic stress resistance, general performance and disease resistance.     

Variation in the absorption of macromolecular proteins in larvae of the sea bassDicentrarchus labrax during transition to the exotrophic phase

Absorption of proteins by pinocytosis was studied in the postvalvular intestine of larvae of the sea bassDicentrarchus labrax during transition to the exotrophic phase. Comparison of specimens ranging in age from 4 or 5 d (age of mouth opening) to 25 d (period of strictly exotrophic feeding) made it possible to determine the morphofunctional development of enterocytes when fish are fed onArtemia sp. (Brazil strain). The pinocytotic activity of enterocytes was weak at the time of mouth opening, but intensified rapidly. After the first feed (7 to 8 d), enterocytes were capable of absorbing proteins, since they actively incorporated ferritin. At the age of 18 to 19 d, they showed a strong capacity for absorption resulting in the formation of a large supranuclear vacuole by the age of 25 d. Examination of the intestine of larvae fed artificially showed that enterocytes developed little until 18 to 19 d of age, but the presence of supranuclear vacuoles in the cytoplasm nevertheless indicated a capacity for absorption.     

Effect of light intensity on activity and food-searching of larval herring,Clupea harengus: a laboratory study

Larvae of Clupea harengus were reared from spawning herring caught in March 1982 and 1983 in the Firth of Clyde, Scotland. An infra0red observation technique was used to record the behaviour of larval herring both in shallow dishes using a top view and in a tank 2 m deep using a side view. The amount of time larvae spent swimming, which was minimum in complete darkness, increased with increasing light intensity and as the larvae grew. Maximum swimming speeds of feeding larvae were recorded at light intensities between 10 and 100 lux. The presence of food organisms (Artemia sp., Brazilian strain) at light intensities below the feeding threshold (0.1 lux) caused an increase in the proportion of time spent active, but light intensities above the threshold had different effects, depending on developmental stage: larvae of 12 mm increased swimming speed, but 21 mm larvae decreased speed. In the 2 m deep tank in darkness, larvae displayed inactive periods wherein they sank head first, interspersed with periods of upward swimming. As light intensity increased, vertical swimming was replaced by horizontal swimming. These results are discussed with reference to food searching and vertical migration of larval herring in the sea.     

Food selection by laboratory-reared larvae of the scaled sardine Harengula pensacolae (Pisces,Clupeidae) and the bay anchovy Anchoa mitchilli (Pisces,Engraulidae)

Food selection by laboratory-reared larvae of scaled sardines Harengula pensacolae, and bay anchovies Anchoa mitchilli, was compared. Natural plankton was fed to the larvae during the 22 days following hatching. Food levels in the rearing tanks were maintained at an average of 1,600 to 1,800 potential food organisms per liter. Larvae of both species selected as food copepod nauplii, copepodites, and copepods; initial feeding was on organisms of 50 to 75 body width. Larvae of H. pensacolae averaged 4.2 mm in total length at hatching and those of A. mitchilli about 2.0 mm. H. pensacolae larvae grew about 1.0 mm per day and A. mitchilli 0.70 mm per day. The mean number of food organisms in each digestive tract was greater in H. pensacolae than in A. mitchilli, and the difference in number increased as the larvae grew. Average size of food organisms eaten increased for both species with growth, because of selection by the larvae; the average size of copepodites and copepods in digestive tracts increased at a faste rate in H. pensacolae than A. mitchilli. A. mitchilli longer than 8 mm did not eat copepod nauplii.Contribution No. 170, Bureau of Commercial Fisheries Tropical Atlantic Biological laboratory, Miami, Florida 33149, USA.     

Appearance of lipid-absorption capacities in larvae of the sea bassDicentrarchus labrax during transition to the exotrophic phase

The development of intestinal lipid absorption capacities was studied in larvae of the sea bassDicentrarchus labrax from the time of mouth opening to 25 d after hatching, in individuals fed with brine shrimp,Artemia sp. (Brazil commercial strain) or artificial diets. At the time of mouth opening, before the first feed, enterocytes synthesized lipoprotein particles from endogenous lipids. Starting with the first feed, enterocytes absorbed food lipids regardless of the diet, but since these cells have a low capacity for lipoprotein synthesis, they accumulated lipids in the form of free lipid droplets. In larvae fed withArtemia sp., the rapid development of enterocytes during growth was combined with increasingly effective lipoprotein synthesis (becoming even greater than that observed in the adult), starting on Days 18 to 19. Although lipoprotein synthesis and transport were observed in larvae given artificial feed, they showed abnormalities in their intestinal mucosa. Lipid droplets formed in association with the membrane structures of specimens given egg yolk during the first days of feeding, and enterocytes degenerated. In 18 to 19 d-old larvae given artificial feed, intestinal folds disappeared and the constituent cells showed limited differentiation. In the last-mentioned individuals, the abnormalities appeared to be related to malnutrition resulting from inadequacy of the food offered, which led to total mortality of larvae, beginning on Day 20.